The long-term goal of this project is to develop a regulatory T cell (Treg)-based approach for the induction of donor-specific immunologic tolerance in liver transplant recipients. Liver transplantation can be life-saving therapy for live failure. However, the maintenance of the transplanted liver requires continuous immunosuppression to prevent rejection by the host immune system. Although ongoing refinement of immunosuppression regimens has substantially reduced the incidence of acute rejection after transplantation, long-term outcomes have stagnated partly due to morbidity and mortality associated with immunosuppression. Therefore, a main focus of research has been to promote tolerance to transplanted livers so that immunosuppression can be minimized or completely withdrawn. In the past decade, we have learned that tolerance in organ transplantation is linked to the development and persistence of Tregs. In multiple preclinical models, therapeutic administration of Tregs has proven efficacy in controlling allograft rejection and inducing donor-specific tolerance. Alloantigen-specific Treg are more effective and potentially safer than non-specific Treg by offering targeted therapy instead of indiscriminate regulation. A key point in Treg-based regimens in the transplant setting is that, because of the exceptionally high frequency of donor-reactive T cells, debulking of the host alloreactive repertoire and adjunct immunosuppression are needed to create a more favorable setting for Tregs to control alloimmunity and to ensure long-term graft tolerance. We aim to translate these basic and clinical findings into a practical and effective clinical protocol. We plan to test the ue of donor- specific Tregs in the context of a Treg-supportive immunosuppression regimen as an approach to induce liver transplant tolerance. As a first step, we propose to conduct an open-label, single center, phase I, dose escalation trial to determine the safety of administering a single escalating dose of donor-specific Tregs in liver transplant patients. We will perform mechanistic analyses of the study patients to assess the impact of the Treg therapy on recipient's immune reactivity to the donor. The R34 grant will allow the investigators to finalize all facets of the clinical trial protocol, Treg manufacturing, and concomitant mechanistic studies, along with securing Investigational New Drug and Institutional Review Board approvals for the trial. To our knowledge, this proposal represents the first application of Tregs in solid organ transplantation to induce graft tolerance. It is strongly aligned with NIAID's goal to evaluate approaches that include tolerogenic, anti-inflammatory, and immunomodulatory strategies to treat and prevent immune-mediated diseases.